© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 1480-1491 doi:10.1242/dev.117960
RESEARCH ARTICLE
Drosophila Vps4 promotes Epidermal growth factor receptor
signaling independently of its role in receptor degradation
ABSTRACT
Endocytic trafficking of signaling receptors is an important
mechanism for limiting signal duration. Components of the
Endosomal Sorting Complexes Required for Transport (ESCRT),
which target ubiquitylated receptors to intra-lumenal vesicles (ILVs)
of multivesicular bodies, are thought to terminate signaling by the
epidermal growth factor receptor (EGFR) and direct it for lysosomal
degradation. In a genetic screen for mutations that affect Drosophila
eye development, we identified an allele of Vacuolar protein sorting 4
(Vps4), which encodes an AAA ATPase that interacts with the
ESCRT-III complex to drive the final step of ILV formation.
Photoreceptors are largely absent from Vps4 mutant clones in the
eye disc, and even when cell death is genetically prevented, the
mutant R8 photoreceptors that develop fail to recruit surrounding
cells to differentiate as R1-R7 photoreceptors. This recruitment
requires EGFR signaling, suggesting that loss of Vps4 disrupts the
EGFR pathway. In imaginal disc cells mutant for Vps4, EGFR and
other receptors accumulate in endosomes and EGFR target genes
are not expressed; epistasis experiments place the function of Vps4
at the level of the receptor. Surprisingly, Vps4 is required for EGFR
signaling even in the absence of Shibire, the Dynamin that
internalizes EGFR from the plasma membrane. In ovarian follicle
cells, in contrast, Vps4 does not affect EGFR signaling, although it is
still essential for receptor degradation. Taken together, these
findings indicate that Vps4 can promote EGFR activity through an
endocytosis-independent mechanism.
KEY WORDS: Vps4, Endocytosis, EGF receptor, Dynamin, Signaling
INTRODUCTION
Endocytosis plays a dual role in signaling by many receptors; it is the
route that leads to receptor degradation, but it can also alter the level
of signaling activity by controlling receptor or ligand processing,
recycling, localization or interaction with downstream components
(Andersson, 2012; Callejo et al., 2011; Musse et al., 2012; Shilo and
Schejter, 2011; Ueno et al., 2011). Activation of many receptors
induces their ubiquitylation, internalization into early endosomes,
sorting to multivesicular bodies (MVBs), where they are segregated
from the cytoplasm, and ultimately lysosomal degradation (Piper and
Lehner, 2011). The GTPase Dynamin catalyzes fission of Clathrincoated endocytic vesicles from the plasma membrane (Schmid and
Frolov, 2011). The subsequent sorting process is mediated by the
Endosomal Sorting Complexes Required for Transport (ESCRT)
Kimmel Center for Biology and Medicine of the Skirball Institute and Department of
Cell Biology, NYU School of Medicine, 540 First Avenue, New York, NY 10016, USA.
*Present address: Institut Jacques Monod, UMR7592 CNRS/Université Paris
Diderot, 15 rue Hé lène Brion, Paris 75205, Cedex 13, France.
‡
Author for correspondence ( [email protected])
Received 17 September 2014; Accepted 20 February 2015
1480
machinery (Hanson and Cashikar, 2012). ESCRT-0, which consists
of the Hepatocyte growth factor regulated tyrosine kinase substrate
(Hrs) and Signal transduction adaptor molecule (Stam) subunits,
binds to and clusters ubiquitylated receptors. ESCRT-0 is recruited to
endosomal membranes through interactions between Hrs and
phosphatidylinositol 3-phosphate. It then recruits the four-subunit
ESCRT-I complex, which brings in ESCRT-II, the substrate for
assembly of ESCRT-III. The ESCRT-III subunit Vacuolar proteinsorting-associated protein 32 (Vps32) polymerizes into filaments
that interact with Vps24 and Vps2 to deform the endosome
membrane and promote budding of cargo-containing intra-lumenal
vesicles (ILVs) (Henne et al., 2012; Wollert and Hurley, 2010). Vps2
also recruits the ATPase associated with a variety of cellular activities
(AAA) protein Vps4, the only energy-utilizing ESCRT component
(Hanson and Cashikar, 2012; Raiborg and Stenmark, 2009). Active
Vps4 forms a hexameric complex that disassembles ESCRT-III,
allowing recycling of its components, and also plays an active role in
scission of the vesicle neck (Adell et al., 2014; Cashikar et al., 2014;
Lata et al., 2008; Monroe et al., 2014; Mueller et al., 2012). In
addition to their endocytic functions, ESCRT proteins, including
Vps4, are required for cytokinesis, viral budding, protecting viral
genomes from degradation, exosome secretion, receptor shedding on
microvesicles, assembly of nuclear pore complexes, cholesterol
transport and plasma membrane wound repair (Barajas et al., 2014;
Choudhuri et al., 2014; Du et al., 2013; de Gassart et al., 2004;
Jimenez et al., 2014; Morita, 2012; Nabhan et al., 2012; Tang, 2012;
Webster et al., 2014).
The effect of endocytosis on signaling by the epidermal growth
factor receptor (EGFR) is complex. Blocking EGFR internalization
by removing Dynamin prevents its degradation, enhancing some
downstream signaling events, but other aspects of EGFR signal
transduction require an endosomal localization for the receptor
(Jones and Rappoport, 2014; Legent et al., 2012; Miura et al., 2008;
Teis et al., 2006; Vieira et al., 1996). Internalized EGFR can be either
degraded or recycled to the plasma membrane, a choice that depends
on the concentration and nature of the ligand, as ligands that remain
bound in acidic late endosomes promote more extensive receptor
ubiquitylation (Eden et al., 2012; French et al., 1995; Roepstorff
et al., 2009; Sigismund et al., 2005). Recycling can alter the
distribution of the receptor on the plasma membrane, controlling its
exposure to ligands (Assaker et al., 2010; Jékely et al., 2005; Stetak
et al., 2006; Vermeer et al., 2003). In Drosophila, EGFR targeted to
the degradation pathway remains active on endosomes, as signaling
is increased by ESCRT mutations that trap EGFR in the endocytic
pathway (Vaccari et al., 2009). However, the transcriptional response
in cultured mammalian cells depends primarily on EGFR activity at
the plasma membrane (Brankatschk et al., 2012; Sousa et al., 2012).
Observed effects on EGFR signaling could vary depending both
on the stage at which endocytic sorting is blocked and the
cellular context examined (Babst et al., 2000; Bache et al., 2006;
Chanut-Delalande et al., 2010; Lloyd et al., 2002; Miura et al., 2008).
DEVELOPMENT
Kevin Legent*, Hui Hua Liu and Jessica E. Treisman‡
RESEARCH ARTICLE
Development (2015) 142, 1480-1491 doi:10.1242/dev.117960
In the Drosophila retina, EGFR signaling is essential for the
differentiation of all photoreceptors except for R8, the first to
differentiate in each ommatidium (Freeman, 1996). Adult eye
phenotypes thus provide a rapid screen for new regulators of the
EGFR pathway (Legent et al., 2012; Miura et al., 2008; Roignant
et al., 2006; Roignant and Treisman, 2010). Here, we report that a
mutation in Vps4 disrupts EGFR signal transduction as well as the
transduction of other signaling pathways. In Vps4 mutant cells in the
eye disc, EGFR accumulates in endosomes but is unable to signal,
resulting in the failure of non-R8 photoreceptors to differentiate.
Interestingly, removing the Dynamin encoded by shibire does not
restore EGFR signaling in the absence of Vps4, suggesting a new
role for Vps4 upstream of EGFR internalization. Vps4 mutant
ovarian follicle cells accumulate endocytosed EGFR, but show
normal expression of EGFR target genes, indicating that defects in
EGFR signaling in imaginal discs are not a consequence of its
sequestration in the endocytic pathway. Taken together, these results
suggest that Vps4 promotes EGFR activation independently of its
effects on endocytosis.
RESULTS
Vps4 is required for R8 survival and R1-R7 differentiation
Fig. 1. Vps4 is required for photoreceptor differentiation. (A) Vps43B1
mutant clones in third-instar eye discs, marked by the absence of GFP (A′,
green in A″), lack photoreceptors stained with anti-Elav (A, magenta in A″).
Anterior is to the left in this and all subsequent figures. (B) Diagram of the Vps4
gene and encoded protein indicating that the 3B1 mutation changes glutamic
acid 209 in the large AAA ATPase domain into a lysine residue. MIT,
microtubule interacting and trafficking domain. The ATPase domain is
subdivided into large, small and beta domains. (C-E) Eye discs expressing
GFP (green) alone (C) or together with HA-tagged wild-type Vps4 (D,E), within
Vps43B1 mutant clones. HA::Vps4 (stained for HA, red in E) rescues the
differentiation of photoreceptors labeled with Elav (blue) including R8, which is
marked by Sens (red in C,D).
The rescue of cell death observed in the absence of Dronc allowed
us to examine early markers of photoreceptor differentiation in Vps4
mutant cells. In each ommatidium, R8, marked by expression of
Senseless (Sens) (Frankfort et al., 2001), is the first photoreceptor to
differentiate. R8 cells are singled out of an anterior stripe of
proneural precursors expressing Atonal (Ato) (Jarman et al., 1993).
Only a few Sens-positive R8 cells were observed in anterior regions
of Vps43B1 mutant clones (Fig. 2C). However, preventing cell death
by removing Dronc rescued many R8 photoreceptors (Fig. 2D),
indicating that Vps4 is required for R8 survival. R8 induces EGFR
activation in surrounding cells to promote their differentiation into
R1-R7 photoreceptors (Freeman, 1997; Tio et al., 1994). In Vps4
clones, most rescued R8 cells failed to recruit any Elav-positive
neighboring cells (Fig. 2E,F). The absence of R1-R7 photoreceptors
is thus not a secondary consequence of cell death, but results from a
failure to transduce signals from R8.
Vps4 mutant cells accumulate inactive signaling receptors
Mutations in Tumor susceptibility gene 101 (TSG101) of the
ESCRT-I complex and Vps25 of the ESCRT-II complex cause
accumulation of the receptor Notch (N), which drives excessive
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DEVELOPMENT
In a mosaic screen of the X chromosome (Legent et al., 2012), we
recovered a mutation that prevents photoreceptor differentiation. In
eye imaginal discs, clones of 3B1 mutant cells showed a cellautonomous lack of expression of the pan-neuronal marker Elav
(Fig. 1A), normally expressed by differentiating photoreceptors
(Robinow and White, 1991). Genetic mapping and sequencing
revealed that 3B1 was a missense mutation in the Vps4 gene. 3B1
transforms glutamate 209, which is adjacent to the first central pore
motif of the AAA domain (Scott et al., 2005), into a lysine residue
(Fig. 1B), a charge reversal that would probably disrupt protein
folding. 3B1 failed to complement the lethality of Vps4Δ7b, a small
deficiency that covers Vps4 and its flanking sequences (Rodahl
et al., 2009a). Expression of an HA-tagged wild-type Vps4 cDNA in
3B1 clones fully rescued photoreceptor differentiation (Fig. 1C-E).
Additionally, 98% (n=106) of hemizygous 3B1 males were rescued
to viability by tubulin-GAL4-driven ubiquitous expression of UASHA::Vps4. These results confirm that the phenotypes observed in
3B1 mutants are due to loss of Vps4 function.
The absence of photoreceptors in Vps4 mutant clones could be
due to either failure of differentiation or cell death. We observed that
Vps43B1 mutant clones in third-instar eye discs underwent massive
apoptosis, as indicated by their pyknotic nuclei and high levels of
activated effector caspases (Fig. 2A). Both features were largely
rescued in the absence of the initiator caspase Dronc (Steller, 2008)
(Fig. 2B). Signaling pathways converging on c-Jun N-terminal
Kinase (JNK) have been implicated in the regulation of programmed
cell death in various contexts (Dhanasekaran and Reddy, 2008). As
previously reported for Vps4 RNAi expression (Rodahl et al.,
2009a), we found that Vps43B1 clones misexpressed the JNK
transcriptional target puckered ( puc)-lacZ (Martin-Blanco et al.,
1998) (supplementary material Fig. S1A). To assess whether ectopic
JNK signaling is instructive in photoreceptor cell death, we created
clones mutant for both Vps4 and hemipterous (hep), which encodes
the upstream activating kinase for JNK (Glise et al., 1995). However,
removing hep did not restore photoreceptor differentiation or prevent
caspase activation (supplementary material Fig. S1B-F). Similarly,
inhibiting JNK signaling by overexpressing the JNK phosphatase
Puc failed to rescue the Vps4 phenotypes (supplementary material
Fig. S1G,H). JNK activity is thus not the primary driver of apoptosis
in Vps4 mutant cells in the eye disc.
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Development (2015) 142, 1480-1491 doi:10.1242/dev.117960
signaling was reduced in Vps4 mutant cells, as visualized by lower
levels of the transcriptional reporter E(spl)mβ-CD2 (de Celis et al.,
1998; Moberg et al., 2005) in the eye disc (Fig. 3C). We also
investigated the requirement for Vps4 in N signaling during wing
development. As Vps4 clones in the wing disc survived poorly
(supplementary material Fig. S2), we induced them in a Dronc
background, and again observed accumulation of N in large puncta
(Fig. 3D). In the wing disc, N is activated in a stripe of cells along
the dorsal-ventral (D-V) boundary, where it induces the
transcription of cut (ct) (de Celis et al., 1996). Ct expression was
lost in Vps4 clones (Fig. 3E), confirming that, in contrast to moreupstream ESCRT mutants, Vps4 promotes N signal transduction.
We next investigated whether other signaling pathways were
similarly affected. In the wing disc, Wingless (Wg) secreted from
both the D-V boundary and the hinge surrounding the wing pouch
acts at a long range to organize wing growth and patterning
(Neumann and Cohen, 1997). Like N and Dl, Wg accumulated in
large punctate structures in Vps4 mutant cells (Fig. 3F). In both the
wing and eye discs, Wg only accumulated in mutant cells within or
close to its endogenous domain of expression, which is marked by
the reporter wg-lacZ (Fig. 3H-I). wg-lacZ was unaffected by the loss
of Vps4 (Fig. 3H), indicating that Wg protein accumulation results
from internalization into receiving cells rather than increased
transcription. Vps4 clones showed reduced expression of the Wg
target genes sens in the wing disc (Fig. 3G) (Parker et al., 2002) and
dachsous-lacZ in the eye disc (Yang et al., 2002) (data not shown),
demonstrating that normal Wg signaling requires Vps4.
In contrast to the reduction in N and Wg signaling, we observed
increased levels of the second messenger phosphorylated Smad
( pSmad) (Tanimoto et al., 2000) in Vps4 mutant clones near the
domain of expression of the Bone Morphogenetic Protein family
member Decapentaplegic (Dpp) in the eye disc (Fig. 3J), suggesting
that Vps4 mutant cells are able to transduce some signals and that, in
contrast to its effects on other pathways, Vps4 negatively regulates
signaling by Dpp. Taken together, our results indicate that unlike
more-upstream ESCRT members, Vps4 is required for the
transduction of several signaling pathways.
Fig. 2. Vps4 is independently required for cell survival and R1-R7
differentiation. (A-F) Eye discs carrying Vps43B1 mutant clones marked by the
absence of GFP (green), in a wild-type (A,C) or DroncI29 (drcI29) (B,D-F)
background. Vps43B1 clones display elevated levels of activated Caspase 3
(A, magenta in A′) and pyknotic nuclei stained with DAPI (A″), which are greatly
reduced in DroncI29 homozygotes (B). Very few Sens-positive R8 cells (C, blue
in C′,C″) differentiate in Vps43B1 clones (arrowheads). Expression of Ato (red)
in anterior proneural precursors is decreased but not abolished. (D) A
substantial rescue of differentiated R8 photoreceptors stained with Sens (blue)
and Ato (red) is observed in DroncI29 homozygotes (arrowheads). (E,F)
Vps43B1 clones in DroncI29 discs stained for Elav (E, blue in E′,E″) and Sens
(red). (F) An enlargement of the region boxed in E. Within the Vps43B1 clone
(outlined), R8 cells are not surrounded by Elav-positive photoreceptors
(arrowheads).
signaling, leading to nonautonomous overproliferation (Moberg
et al., 2005; Vaccari and Bilder, 2005). Similarly, we found that
Vps4 mutant cells accumulated very high levels of both N and its
ligand Delta (Dl) in punctate structures (Fig. 3A,B). However,
unlike the upstream ESCRT mutants TSG101 and Vps25, N
1482
The effect of receptor internalization and endocytic processing on
EGFR signaling remains controversial. Although some studies have
demonstrated that it contributes to signal termination through
lysosomal EGFR degradation (Bache et al., 2006; Razi and Futter,
2006), other results argue that it enables receptor signaling from
intracellular organelles (Miaczynska et al., 2004; Shilo and Schejter,
2011). Vps4 clones in Dronc mutant eye discs resemble clones
lacking positive intracellular effectors of EGFR transduction, in
which R8 cells fail to recruit additional photoreceptors (Legent
et al., 2012). We found that the expression of lacZ reporters for two
direct transcriptional targets of the EGFR pathway in R1-R7, argos
(aos) (Golembo et al., 1996) and hedgehog (hh) (Rogers et al.,
2005), was downregulated in Vps4 mutant clones (Fig. 4A,B).
Importantly, restoring R8 survival by removing Dronc did not
rescue aos expression in surrounding cells (Fig. 4C). In the wing
disc, EGFR signaling controls both the expression of aos in vein
primordia (Sturtevant et al., 1993), and the expression of mirror
(mirr) in the notum primordium (Zecca and Struhl, 2002).
Consistent with a requirement for Vps4 in EGFR signaling, Vps4
mutant cells displayed a reduced expression of aos-lacZ and
mirr-lacZ (Fig. 4E,F).
Despite the lack of EGFR target gene expression, EGFR protein
levels were increased in Vps4 mutant cells (Fig. 4D,G,H).
DEVELOPMENT
Vps4 is required for EGFR signaling
RESEARCH ARTICLE
Development (2015) 142, 1480-1491 doi:10.1242/dev.117960
Fig. 3. Loss of Vps4 affects multiple signaling pathways. Vps43B1
clones marked by the absence of GFP (green) in eye discs (A-C,I-J) or
in DroncI29 (drcI29) homozygous wing discs (D-H). (A,B) Mutant cells
accumulate high levels of N (A″, magenta in A,A′) and Dl (B″, magenta
in B,B′) in large puncta (boxed regions are enlarged in A′,A″,B′,B″).
(C) Expression of the Notch transcriptional reporter E(spl)-mβ-CD2
(C, magenta in C′) is decreased in Vps4 clones. (D) Mutant cells
accumulate large N-positive puncta (D″, magenta in D,D′, the boxed
region is enlarged in D′,D″). (E) The Notch transcriptional target Ct
(E,E″, magenta in E′), normally expressed at the boundary between
the dorsal (d) and ventral (v) compartments of the wing pouch, is lost in
Vps4 clones (the boxed region is enlarged in E′,E″). (F) Wg
(F″, magenta in F,F′) also accumulates in large puncta that span the
depth of the cell in Vps4 clones (arrowheads; the boxed region is
enlarged in F′,F″; xz sections are shown below). (G) The Wg target
Sens (red) is expressed in two stripes (arrowheads) along the D-V
boundary of the wing pouch, but is lost from Vps4 clones, despite
accumulation of Wg (blue). The boxed region is enlarged in G′,G″.
(H) Ectopic Wg (red) is observed in Vps4 clones in the vicinity of Wgproducing cells (open arrowheads), marked by wg-lacZ (blue), but not
in more distant clones (filled arrowheads). The boxed region is
enlarged in H′,H″. (I) In eye discs, Wg puncta (I′, magenta in I) are
observed in clones along the lateral margins (open arrowhead) but not
in more medial clones (filled arrowhead). (J) Vps4 clones display
elevated levels of pSmad (J′, magenta in J) in the region of the
morphogenetic furrow (arrowheads).
EGFR signaling is not dependent on the fate or epithelial
organization of imaginal disc cells.
Vps4 acts at the level of EGFR activation
In order to determine at which step Vps4 influences EGFR signal
transduction, we performed epistasis experiments. In the eye disc, Spi
secreted by R8 is the primary ligand for EGFR in R1-R7 (Freeman,
1994; Tio et al., 1994). Its binding triggers receptor dimerization and
activation by autophosphorylation, and subsequent recruitment of
enzymes and adaptor proteins promotes conversion of Ras (also
known as Ras85D) into its GTP-bound form. This small GTPase
initiates a kinase cascade by activating Raf (also known as Pole hole),
which phosphorylates MEK (also known as Dsor1), which in turn
phosphorylates MAPK. Phosphorylated MAPK enters the nucleus
and phosphorylates specific transcription factors to regulate target
gene expression (Shilo, 2003). During eye development, expression
of a secreted form of Spi (sSpi) (Schweitzer et al., 1995) or
constitutively active forms of EGFR (Queenan et al., 1997) or Ras
(Karim and Rubin, 1998) ectopically activates EGFR signaling and
promotes the differentiation of extra photoreceptors (Fig. 5A-C).
Expression of sSpi did not rescue photoreceptor differentiation in
Vps4 mutant cells, although it triggered ectopic photoreceptor
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DEVELOPMENT
Intracellular EGFR colocalized with N in punctate structures, many
of which also expressed the early endosomal markers Hrs, Rab5 and
Syntaxin 7 (Syx7) (Lloyd et al., 2002; Lu and Bilder, 2005; Russell
et al., 2012) (Fig. 4D,G,H; supplementary material Fig. S3A). Little
colocalization was observed with markers for recycling endosomes
(Rab11), the Golgi (Lava lamp; Lva) or secretory vesicles (Sec15)
(Langevin et al., 2005; Sisson et al., 2000) (supplementary material
Fig. S3B-D), arguing that the EGFR accumulation is in enlarged
endosomes, known as class E compartments in yeast (Russell et al.,
2012). Consistent with this view, loss of the ESCRT-I subunit
TSG101 caused a similar accumulation of EGFR in punctate
structures expressing Hrs (supplementary material Fig. S3F).
S2 cells stably expressing the EGFR (D2F cells) have been used
to study EGFR signaling mechanisms (Schweitzer et al., 1995).
Treatment of these cells with a purified secreted form of the EGFR
ligand Spitz (Spi) (Miura et al., 2006) resulted in increased
production and secretion of Aos protein (Fig. 4I). However, RNAimediated knockdown of Vps4 (supplementary material Fig. S4A)
strongly reduced this response (Fig. 4I). Vps4 RNAi also reduced
the phosphorylation of the downstream effector MAPK (also known
as ERK and Rolled) in cells treated with Spi (supplementary
material Fig. S4B). These results indicate that the effect of Vps4 on
RESEARCH ARTICLE
Development (2015) 142, 1480-1491 doi:10.1242/dev.117960
Fig. 4. Vps4 is required for EGFR signaling. Eye discs (A-D,G-H) or wing discs (E,F) in which Vps43B1 mutant clones are marked by the absence of GFP (green,
A-D,G-H) or RFP (green, E,F), in a wild-type (A-B,E-F) or DroncI29 (drcI29) homozygous (C-D,G-H) background. Vps43B1 clones lose expression of the EGFR
transcriptional targets aos-lacZ (A, magenta in A′) and hh-lacZ (B, magenta in B′). (C) R8 cells marked by Sens (blue) in Vps4 clones in Dronc homozygotes
fail to induce aos-lacZ (C, red in C′) in surrounding cells. (D) EGFR (D, red in D″,D‴) and N (D′, blue in D″,D‴) accumulate and colocalize in punctate
structures in Vps4 mutant cells (xz sections are shown below). (E) aos-lacZ (E′, magenta in E,E‴) is normally expressed along presumptive veins but is lost in
Vps43B1 clones (arrowheads), which accumulate high levels of N (E″, green in E‴). (F) In the notum, expression of the EGFR transcriptional target mirr-lacZ
(F′, magenta in F,F″) is decreased in Vps4 mutant cells. The boxed region is enlarged in F′,F″. (G,H) In Vps4 mutant cells, punctate accumulations of EGFR
(G,H, blue in G″,H″) colocalize extensively with Hrs (H′, red in H″) and partially with Rab5 (G′, red in G″). Both xy and xz sections are shown. (I) Western
blots of Aos in the culture media and β-tubulin in the cell lysates of triplicate samples of D2F cells treated with lacZ or Vps4 dsRNA as indicated, and exposed (+) or
not exposed (−) to purified sSpi for 16 h. Vps4 knockdown strongly reduces Aos production.
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the cell death observed in Vps4 mutant clones. Indeed, although
activated EGFR did not prevent caspase activation in Vps4 cells
(Fig. 5H-J), activated Ras abolished apoptosis (Fig. 5K,L). These
results demonstrate that, rather than ectopic JNK signaling
(supplementary material Fig. S1) (Rodahl et al., 2009a), impaired
EGFR signal transduction is the primary reason for the death of
Vps4 mutant cells.
Vps4 functions upstream of EGFR endocytosis
Because the effect of Vps4 on EGFR signaling was opposite to the
negative effect of other ESCRT components (Vaccari et al., 2009)
(see supplementary material Fig. S3E), we wanted to test whether
Vps4 acted during endocytosis to affect EGFR signaling. We
therefore generated double mutant clones for Vps4 and shibire (shi),
which encodes Dynamin, a GTPase required to internalize EGFR
and other receptors from the plasma membrane (Henriksen et al.,
2013; Sousa et al., 2012; Windler and Bilder, 2010). shi7C7 mutant
cells have enhanced EGFR signaling, leading to increased
photoreceptor differentiation (Legent et al., 2012) (Fig. 6A). The
ESCRT-0 component Hrs has a positive role in EGFR signaling and
photoreceptor differentiation in the eye disc (Miura et al., 2008). As
expected, given that Dynamin acts prior to Hrs in endocytosis, shi
DEVELOPMENT
differentiation in wild-type cells surrounding the clone, indicating that
Vps4 is not necessary for Spi secretion (Fig. 5D). The same result was
obtained by overexpressing Rhomboid, a protease that activates
endogenous Spi (Lee et al., 2001) (data not shown). Interestingly, a
constitutively dimeric form of EGFR showed partial activity in Vps4
mutant cells; although photoreceptors still failed to differentiate
within Vps4 mutant clones expressing activated EGFR, surrounding
cells showed premature differentiation (Fig. 5E), probably due to hh
expression induced within the clone (supplementary material Fig. S5).
Expression of activated Ras was sufficient to bypass the requirement
for Vps4 in EGFR transduction and promote the differentiation of
extra photoreceptors both within and surrounding the clone (Fig. 5F).
Similar epistasis experiments in the wing disc, using aos-lacZ to
monitor pathway activation, showed that activated EGFR promoted
strong ectopic aos expression in wild-type but not Vps4 mutant cells,
whereas activated Ras could upregulate aos-lacZ even in the absence
of Vps4 (supplementary material Fig. S6). The activity of Vps4 is thus
required in the receiving cell, upstream of Ras activation but
downstream or at the level of EGFR activation (Fig. 5G).
Given that the EGFR pathway is required for cell survival in the
eye disc (Halfar et al., 2001; Yang and Baker, 2003), we wondered
whether restoring signaling downstream of the receptor could rescue
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Development (2015) 142, 1480-1491 doi:10.1242/dev.117960
mutant cells still showed increased photoreceptor differentiation in
Hrs mutant eye discs, although loss of Hrs reduced photoreceptor
differentiation in regions wild-type for shi (Miura et al., 2008)
(Fig. 6B). Surprisingly, however, shi7C7 Vps43B1 double mutants
showed the Vps4 phenotype of missing photoreceptors and loss of
aos-lacZ expression, even in a Dronc background in which cell
death was prevented (Fig. 6C,D). Vps4 was similarly epistatic to shi
in clones expressing a dominant-negative thermo-sensitive allele of
shi (Kitamoto, 2001) (Fig. 6E,F). As the molecular nature of the
shi7C7 allele is unknown, we repeated the epistasis analysis with the
previously described null allele shiFL54 (Windler and Bilder, 2010).
shiFL54 Vps43B1 double mutant cells also failed to differentiate as
photoreceptors, although removing shi prevented the endosomal
accumulation of EGFR in these cells (Fig. 6G,H). These results are
not consistent with an effect of Vps4 only on internalized EGFR,
and suggest that Vps4 can influence EGFR function prior to its
endocytosis.
One possibility is that Vps4 might promote EGFR trafficking to
the plasma membrane. To investigate this, we made use of the
ovarian model system, in which the ligand-producing and
responding cells can be distinguished. During egg chamber
development, the EGFR ligand Gurken (Grk) is secreted from the
oocyte and internalized into first posterior and later dorsal anterior
follicle cells to specify their fates (Chang et al., 2008; Nilson and
Schüpbach, 1998). This internalization requires both shi and Egfr
(Chang et al., 2008), indicating that it is mediated by binding to
surface EGFR and subsequent endocytosis. We found that clones of
follicle cells mutant for Vps4 were still able to internalize Grk, and
in fact accumulated abnormally high levels of Grk that colocalized
with internalized EGFR in punctate structures (Fig. 7A). EGFR
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DEVELOPMENT
Fig. 5. Vps4 acts at the level of the EGFR.
(A-F,H-L) Eye discs with clones coexpressing
GFP (green) with activated pathway
components, stained for Elav (A-F and blue in
A′-F′,H-L). Sens is shown in red in A′-F′.
Expression of the constitutively active
proteins encoded by sSpi (A), EGFRλtop (B) or
RasV12 (C) in wild-type clones induces
excessive photoreceptor differentiation. In
Vps43B1 clones, expression of sSpi (D)
induces extra photoreceptor differentiation in
wild-type cells surrounding the clone, but has
no effect on the mutant cells. EGFRλtop
expression in Vps4 clones (E) does not
rescue photoreceptor differentiation within the
clones, but induces ectopic photoreceptors in
surrounding wild-type tissue. In contrast,
RasV12 can induce photoreceptor
differentiation within Vps4 mutant clones (F).
(G) A simplified diagram of the EGFR pathway
showing that Vps4 acts upstream of Ras
activation to promote photoreceptor
differentiation and cell survival. (H-L)
Activated Caspase 3 is stained in red. Control
EGFRλtop (I) or RasV12 (K) expressing clones
do not induce cell death. The caspase
activation observed in Vps43B1 clones (H) is
rescued by UAS-RasV12 (L) but not UASEGFRλtop (J).
RESEARCH ARTICLE
Development (2015) 142, 1480-1491 doi:10.1242/dev.117960
must therefore be present on the surface of Vps4 mutant follicle
cells, allowing Grk reception. Surprisingly, Vps4 was not required
for EGFR signaling in these cells. In this system, EGFR signaling
acts by preventing nuclear localization of the transcriptional
repressor Capicua (Cic) (Astigarraga et al., 2007). Cic represses
the expression of mirr (Atkey et al., 2006), which encodes a
transcription factor that represses the ventral determinant pipe
(Andreu et al., 2012). In dorsal follicle cells mutant for Vps4, a mirrlacZ reporter was expressed normally, and no ectopic pipe-lacZ or
nuclear localization of Cic was observed (Fig. 7B,C). The large
clone size also indicated normal survival of these mutant cells.
Notch signaling was likewise unaffected in Vps4 mutant follicle
cells. We saw no loss of its target gene hindsight (hnt; FlyBase –
peb) or misexpression of ct, a gene repressed by Hnt (Sun and Deng,
2007), and mitotic cells marked by phosphorylated histone H3 were
not observed in egg chambers older than stage 6 (Fig. 7D,E), despite
strong intracellular accumulation of Notch protein (Fig. 7F). These
results show that trapping of the EGFR with its ligand in the
endocytic pathway is not sufficient to block EGFR signaling. Vps4
thus appears to promote EGFR signaling by a new non-endocytic
and cell type-dependent mechanism, which could also be applicable
to Notch and other receptors.
DISCUSSION
Vps4 affects multiple signaling pathways
We describe here a point mutation that specifically disrupts the
function of Drosophila Vps4. A previous study used a deletion
1486
allele that unlike our mutation, could not be rescued by a wild-type
Vps4 construct, and thus might also disrupt one or both of the
neighboring genes (Rodahl et al., 2009a). We confirmed previous
findings that loss of Vps4 results in JNK activation and apoptosis
(Rodahl et al., 2009a). However, we found that blocking JNK
activity did not prevent cell death or restore photoreceptor
differentiation, indicating that JNK is not the primary driver of
these effects of Vps4 mutations. In contrast, activating EGFR
signaling downstream of the receptor was sufficient to restore
survival of R8 cells and differentiation of R1-R7, highlighting this
pathway as central to the function of Vps4 in eye development.
As expected, given the known role for Vps4 and other ESCRT
proteins in targeting receptors for lysosomal degradation (Hanson
and Cashikar, 2012), EGFR and other receptors accumulate in
enlarged endosomes in Vps4 mutant cells. However, this
accumulation has distinct effects on their activity; loss of Vps4
reduces EGFR, Notch and Wg signaling, but increases Dpp
signaling. The effect on Wg target genes is consistent with
previous findings that endocytosis and MVBs promote Wnt
signaling by sequestering Glycogen synthase kinase 3β, which
would otherwise inhibit β-catenin (Dobrowolski et al., 2012; Seto
and Bellen, 2006; Taelman et al., 2010). Dpp signaling is also
thought to require endocytosis, because Smads are recruited to
activated TGFβ family receptors by the endosomal protein Smad
anchor for receptor activation (Sara) (Bennett and Alphey, 2002;
Panopoulou et al., 2002; Tsukazaki et al., 1998). As Sara is present
on early endosomes, signaling can be prolonged when progression
DEVELOPMENT
Fig. 6. Vps4 blocks EGFR signaling
upstream of Dynamin-mediated
internalization. All panels show eye
discs with clones marked by the
absence of GFP (green) in A-D,G-H, or
the presence of GFP (E,F). R8 is
stained with Sens (red in A-F).
Photoreceptors are stained with Elav
(A′-C′,E′,F′, blue in A-C,E,F) or aos-lacZ
is stained with anti-β-galactosidase
(D′, blue in D). (A) shi7C7 clones
differentiate excessive photoreceptors.
(B) In an HrsD28/Df(Hrs) mutant disc in
which photoreceptor differentiation is
reduced (open arrowheads), shi7C7
clones still overproduce photoreceptors
(filled arrowheads). (C,D) shi7C7
Vps43B1 double mutant clones in a
DroncI29 (drcI29) mutant background
show loss of photoreceptors other than
R8 and loss of aos-lacZ expression.
(E,F) Clones expressing shits in
otherwise wild-type cells (E) or in
Vps43B1 mutant cells (F). Vps4 is still
required for photoreceptor
differentiation in the presence of this
dominant-negative form of Shi. (G,H)
shiFL54 Vps43B1 clones (outlined in H) in
a DroncI29 mutant background are
stained with Elav (G′, magenta in G) or
EGFR (H′, magenta in H). Clones
mutant for both Vps4 and a shi-null
allele lack photoreceptors but do not
show punctate accumulation of EGFR.
RESEARCH ARTICLE
Development (2015) 142, 1480-1491 doi:10.1242/dev.117960
of the receptor to late endosomes is blocked by loss of the ESCRT-II
subunit Vps25 (Thompson et al., 2005) and perhaps also Vps4.
Vps4 has effects distinct from other ESCRT complex
subunits
Vps4 mutants differ from previously described ESCRT mutations in
their effects on Notch and EGFR signaling. Internalization of these
receptors into the ILVs of MVBs segregates their intracellular
domains from cytoplasmic effectors and should thus terminate
signaling (Piper and Lehner, 2011). This model is consistent with
the excessive Notch and EGFR signaling observed in eye and wing
discs in the absence of many ESCRT proteins (Aoyama et al., 2013;
Moberg et al., 2005; Rodahl et al., 2009b; Thompson et al., 2005;
Vaccari and Bilder, 2005; Vaccari et al., 2009). This increased
signaling has been attributed to the lack of lysosomal degradation of
these receptors and their continued activity on the endosomal
membrane.
Vps4 mutant cells instead show reduced expression of EGFR and
Notch target genes, despite endosomal accumulation of the
receptors. Vps43B1 cells also fail to induce the non-autonomous
overgrowth that results from misexpression of the Notch target gene
unpaired (FlyBase – outstretched) in cells mutant for other ESCRT
subunits (Thompson et al., 2005; Vaccari and Bilder, 2005). Notch
cleavage and signaling is thought to occur in partially acidified
endosomes and thus to require progression through the endocytic
pathway (Schneider et al., 2013; Vaccari et al., 2010, 2008; Yan
et al., 2009). Although EGFR signaling can occur at the plasma
membrane (Brankatschk et al., 2012; Legent et al., 2012; Sousa
et al., 2012), some studies suggest that receptor progression from
early to late endosomes has a positive effect on signaling (Kim et al.,
2007; Miura et al., 2008; Teis et al., 2006). Loss of Vps4 might
block endocytosis so late in the process of ILV formation that the
intracellular domains of receptors are trapped in nascent ILV buds
and no longer have access to the cytoplasm. Alternatively, an early
1487
DEVELOPMENT
Fig. 7. Vps4 is not required for EGFR or
Notch signaling in follicle cells. All panels
show egg chambers with Vps43B1 mutant
clones in the follicle cells (FC) marked by the
absence of RFP (red in A-E, magenta in F).
OO, oocyte. Posterior is to the right.
Arrowheads indicate representative clones.
(A) Staining for Grk (A′, green in A) and EGFR
(A″, blue in A) reveals that internalized Grk and
EGFR accumulate and colocalize in mutant
follicle cells. (B-C) Dorsal is up. (B) A mutant
clone in follicle cells dorsal to the oocyte shows
normal mirr-lacZ expression (B″, blue in B) and
Grk internalization (B′, green in B). (C) Dorsal
mutant follicle cell clones do not misexpress
pipe-lacZ (C′, green in C) and Cic remains
cytoplasmic, rather than localizing to the
nucleus as seen in the ventral region (C″, blue
in C). (D,E) Hnt (D′, blue in D) is expressed
normally and there is no ectopic phosphohistone H3 ( pH3) staining (D″, green in D,E) in
Vps4 clones. Ct (E′, blue in E) is not
misexpressed in Vps4 clones. Loss of Notch
signaling would cause loss of Hnt and ectopic
Ct and pH3 in egg chambers older than stage
6. (F) Notch (green) accumulates in puncta in
Vps4 clones.
RESEARCH ARTICLE
Vps4 might contribute to EGFR activation
Another possible explanation for the effect of Vps4 on EGFR
signaling is suggested by our finding that loss of shi function does
not restore EGFR signaling to Vps4 mutant cells, even though it
blocks receptor accumulation in the endocytic pathway. Dynamin is
required to internalize mammalian EGFR by both clathrindependent and -independent mechanisms following its activation
with all the ligands tested (Henriksen et al., 2013). In the absence of
Dynamin, prolonged signaling of EGFR on the plasma membrane
should be unaffected by defects in the late stages of endocytosis.
The disruption of EGFR signaling by Vps4 in shi mutant cells thus
suggests that Vps4 has a role upstream of cell surface EGFR
activation that is distinct from its function in endocytic MVBs. This
model is supported by our observation that a constitutively active
form of EGFR can partially rescue Vps4 mutant cells (Fig. 5E). In
addition, although Vps4 affects the progression of EGFR and its
ligand Grk, as well as Notch, through the endocytic pathway in
follicle cells, signaling by both receptors remains intact in these
cells, indicating that the two functions of Vps4 are separable.
The mechanism of this new effect of Vps4 is still unknown. As
Vps4 mutant clones accumulate high levels of intracellular EGFR,
Vps4 is not required for EGFR transcription or translation.
Although some studies have implicated ESCRT-III and Vps4 in
recycling Ras and associated EGFR to the plasma membrane
(Baldys and Raymond, 2009; Tu et al., 2011; Zheng et al., 2012),
Drosophila Ras need not be associated with the membrane to
function in photoreceptor recruitment (Sung et al., 2010), and a
requirement for Vps4 in EGFR recycling would not explain its
effect in the absence of Dynamin-mediated internalization. Vps4
has been reported to transport some newly synthesized proteins to
the plasma membrane via endosomes (Futter et al., 1995; Yoshimori
et al., 2000). However, the ability of Vps4 mutant follicle cells to
internalize Grk indicates that EGFR is present on the plasma
membrane of these cells. It is possible that EGFR is trafficked by
distinct routes in imaginal discs and in follicle cells. Grk is
internalized through the apical domain of follicle cells (Tanentzapf
et al., 2000), whereas active forms of Spi are localized basolaterally
in discs (Steinhauer et al., 2013). A role for Vps4 in targeting the
EGFR to the appropriate membrane domain would, however, not
explain the requirement for Vps4 in cultured S2 cells, which lack
apical-basal polarity. An alternative possibility is that Vps4 affects
EGFR activation independently of its trafficking. Vps4 is involved
in transporting cholesterol to the endoplasmic reticulum (Du et al.,
2013), and could indirectly affect EGFR activation by altering the
composition of membrane rafts (Balbis and Posner, 2010). Loss of
Vps4 in yeast was recently shown to result in the accumulation of
misassembled nuclear pore complexes (Webster et al., 2014);
although it seems unlikely that this would influence EGFR
activation, it supports the existence of previously undescribed
functions for Vps4. Further investigation of the effect of Vps4 on
EGFR activity might define a new molecular mechanism of action
for this versatile protein.
MATERIALS AND METHODS
Drosophila genetics
3B1 is an EMS-induced lethal allele of Vps4 isolated previously (Legent
et al., 2012). Using rescue by X chromosomal duplications followed by
recombination with P-element markers (Zhai et al., 2003), we mapped it to a
1488
0.35 cM interval in 16F1-5. The coding region of Vps4 amplified from
homozygous mutant larvae contained a missense mutation, E209K, that was
absent from the y,w, FRT19A isogenic strain used for the mutagenesis. The
rescuing duplication Dp(1;3)JC153 (R1) was obtained from Alberto Ferrus
(Cajal Institute, Madrid, Spain). hepr75 (Glise et al., 1995) is a deletion of
nucleotides 486 to 1346 that removes the start codon. Other stocks used were
wg-lacZP, puc-lacZE69, UAS-puc2A, HrsD28, Df(Hrs) [Exel6277], aoslacZW11, hh-lacZP30, mirr-lacZcre2 (Bloomington Drosophila Stock Center),
E(spl)mβ-CD2 (de Celis et al., 1998), pipe-lacZ (Andreu et al., 2012),
vps4Δ7b (Rodahl et al., 2009a), DroncI29 (Xu et al., 2005), shi7C7 (Legent
et al., 2012), shiFL54 (Windler and Bilder, 2010), UAS-shits (Kitamoto,
2001), TSG1012 (Moberg et al., 2005), UAS-rhomboid (Lee et al., 2001),
UAS-sSpi (Schweitzer et al., 1995), UAS-EGFRλtop (Queenan et al., 1997)
and UAS-RasV12 (Karim and Rubin, 1998).
Stocks used to generate clones were: (1) w, hsFLP122, P[w+, ubi-GFP],
FRT19A, (2) y,w, hsFLP122, P[w+, ubi-RFP], FRT19A, (3) w, eyFLP1,
tub-GAL80, FRT19A;; tub-GAL4, UAS-CD8::GFP/SM6-TM6B, (4) w,
hsFLP122, tub-GAL80, FRT19A;; tub-GAL4, UAS-CD8::GFP/SM6-TM6B,
(5) w, eyFLP1, P[w+, ubi-GFP], FRT19A; DroncI29/TM6B, (6) w,
hsFLP122, P[w+, ubi-GFP], FRT19A; DroncI29/TM6B and (7) w,
hsFLP122, P[w+, ubi-RFP], FRT19A; DroncI29/TM6B.
Molecular biology
The full-length Vps4 (CG6842) coding region was amplified by PCR from
the GH02678 EST clone (Drosophila Genomics Resource Center) using Pfu
Turbo and cloned into pUAST-HA as an EcoRI-XhoI fragment to generate
pUAS-HA::Vps4. Transgenic flies were generated by Genetic Services, Inc.
Immunohistochemistry
Third-instar eye and wing discs were dissected and stained as described
previously (Legent and Treisman, 2008). Ovaries were stained as described
previously (Miura et al., 2006). Antibodies used were: rabbit anti-βgalactosidase (1:5000, Cappel), chicken anti-GFP (1:500, Aves), mouse
anti-HA (1:100, Covance), mouse anti-CD2 (1:50, Serotec), rabbit antiactive Caspase 3 (1:500, CM1, BD Pharmingen), rabbit anti-Ato (1:5000;
Jarman et al., 1993), guinea pig anti-Sens (1:1000; Nolo et al., 2000),
mouse anti-NECD (1:10), mouse anti-DlECD (1:10), mouse anti-Wg (1:10),
mouse anti-Ct (1:10), mouse anti-Grk (1:10), rat anti-Elav (1:100) (all
Developmental Studies Hybridoma Bank, University of Iowa), rabbit antiEGFRCTER (1:500; Rodrigues et al., 2005), mouse anti-EGFREXT (1:200,
Sigma E2906), rabbit anti-phospho-Smad (1:50, Cell Signaling 9516),
guinea pig anti-Hrs (1:200; Lloyd et al., 2002), rabbit anti-Rab5 (1:50;
Wucherpfennig et al., 2003), rabbit anti-Rab11 (1:1000; Satoh et al., 2005),
chicken anti-Syx7 (1:500; Lu and Bilder, 2005), rabbit anti-Lva (1:5000;
Sisson et al., 2000), guinea pig anti-Sec15 (1:500; Mehta et al., 2005), and
rabbit anti-Cic (1:5000; Astigarraga et al., 2007). Secondary antibodies used
were from Jackson Immunoresearch (1:200) or Molecular Probes (1:1000).
Images were taken on a Leica SP5 confocal microscope. For shits stainings,
larvae were maintained at 31°C for 48 h prior to dissection.
Antibodies used for western blotting were mouse anti-diphosphorylated
ERK (dpERK, 1:2500, Sigma M8159), rabbit anti-ERK (1:20,000, Cell
Signaling 4695), mouse anti-Aos (1:100, Developmental Studies
Hybridoma Bank), and mouse anti-β-Tubulin (1:3000, Covance MMS410P) antibodies.
Cell culture
EGFR-expressing S2 (D2F) cells (Schweitzer et al., 1995) were maintained
in Schneider’s medium supplemented with 10% fetal calf serum and
150 µg/ml G418. Double-stranded RNAs (dsRNAs) were generated using
the MEGAscript T7 and T3 Kits (Ambion) as described previously
(Roignant et al., 2006) and 15 µg dsRNA were used to treat 106 cells per well
for 5 days. EGFR expression was induced for 3 h with 60 µM Cu2SO4. For
dpERK ( pMAPK) western blots, the cells were treated with purified HissSpiCS (Miura et al., 2006) for 10 min, and lysed as described previously
(Miura et al., 2008). For Aos western blots, cells were serum-starved in
medium containing dsRNA, and treated with purified His-sSpiCS for 16 h.
Media were then harvested and the cells were lysed in ice-cold 50 mM
Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100 and protease inhibitors
DEVELOPMENT
block in receptor progression through endocytosis due to failure to
recycle other ESCRT subunits (Babst et al., 1998) could reduce
signaling.
Development (2015) 142, 1480-1491 doi:10.1242/dev.117960
(Roche). Western blotting was carried out as described previously (Miura
et al., 2006). Total RNA was extracted from D2F cells using Trizol
(Invitrogen). RT-PCR was performed on 1 µg of total RNA using the
Invitrogen SuperScript First-Strand Kit. Primer sequences are available on
request.
Acknowledgements
We thank Minrong Ai, Hugo Bellen, David Bilder, Alberto Ferrus, Marcos GonzalezGaitan, Andrew Jarman, Gerardo Jimenez, Daniel Marenda, Don Ready, Aloma
Rodrigues, Hyung Don Ryoo, Benny Shilo, Harald Stenmark, the Drosophila
Genomics Resource Center, the Bloomington Drosophila Stock Center and the
Developmental Studies Hybridoma Bank for fly stocks and reagents. We are grateful
to Antoine Guichet for his support. The manuscript was improved by the critical
comments of Sergio Astigarraga, Justine Oyallon, Josefa Steinhauer and Annabelle
Suisse.
Competing interests
The authors declare no competing or financial interests.
Author contributions
K.L. and J.E.T. designed experiments; K.L., H.H.L. and J.E.T. performed
experiments; K.L. and J.E.T. wrote the manuscript.
Funding
This work was supported by the National Institutes of Health [EY013777 to J.E.T.].
Deposited in PMC for release after 12 months.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.117960/-/DC1
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